A carbohydrate-based mechanism of species recognition in sea urchin fertilization

In the present review, we describe a systematic study of the sulfated polysaccharides from marine invertebrates, which led to the discovery of a carbohydrate-based mechanism of sperm-egg recognition during sea urchin fertilization. We have described unique polymers present in these organisms, especially sulfated fucose-rich compounds found in the egg jelly coat of sea urchins. The polysaccharides have simple, linear structures consisting of repeating units of oligosaccharides. They differ among the various species of sea urchins in specific patterns of sulfation and/or position of the glycosidic linkage within their repeating units. These polysaccharides show species specificity in inducing the acrosome reaction in sea urchin sperm, providing a clear-cut example of a signal transduction event regulated by sulfated polysaccharides. This distinct carbohydrate-mediated mechanism of sperm-egg recognition coexists with the bindin-protein system. Possibly, the genes involved in the biosynthesis of these sulfated fucans did not evolve in concordance with evolutionary distance but underwent a dramatic change near the tip of the Strongylocentrotid tree. Overall, we established a direct causal link between the molecular structure of a sulfated polysaccharide and a cellular physiological event - the induction of the sperm acrosome reaction in sea urchins. Small structural changes modulate an entire system of sperm-egg recognition and species-specific fertilization in sea urchins. We demonstrated that sulfated polysaccharides - in addition to their known function in cell proliferation, development, coagulation, and viral infection - mediate fertilization, and respond to evolutionary mechanisms that lead to species diversity.

Heterofucans from Dictyota menstrualis have anticoagulant activity

Fucan is a term used to denote a family of sulfated L-fucose-rich polysaccharides which are present in the extracellular matrix of brown seaweed and in the egg jelly coat of sea urchins. Plant fucans have several biological activities, including anticoagulant and antithrombotic, related to the structural and chemical composition of polysaccharides. We have extracted sulfated polysaccharides from the brown seaweed Dictyota menstrualis by proteolytic digestion, followed by separation into 5 fractions by sequential acetone precipitation. Gel electrophoresis using 0.05 M 1,3-diaminopropane-acetate buffer, pH 9.0, stained with 0.1% toluidine blue, showed the presence of sulfated polysaccharides in all fractions. The chemical analyses demonstrated that all fractions are composed mainly of fucose, xylose, galactose, uronic acid, and sulfate. The anticoagulant activity of these heterofucans was determined by activated partial thromboplastin time (APTT) using citrate normal human plasma. Only the fucans F1.0v and F1.5v showed anticoagulant activity. To prolong the coagulation time to double the baseline value in the APTT, the required concentration of fucan F1.0v (20 µg/ml) was only 4.88-fold higher than that of the low molecular weight heparin Clexane® (4.1 µg/ml), whereas 80 µg/ml fucan 1.5 was needed to obtain the same effect. For both fucans this effect was abolished by desulfation. These polymers are composed of fucose, xylose, uronic acid, galactose, and sulfate at molar ratios of 1.0:0.8:0.7:0.8:0.4 and 1.0:0.3:0.4:1.5:1.3, respectively. This is the fist report indicating the presence of a heterofucan with higher anticoagulant activity from brown seaweed.

Polysaccharide-rich fraction of Agaricus brasiliensis enhances the candidacidal activity of murine macrophages

A polysaccharide-rich fraction (ATF) of medicinal mushroom Agaricus brasiliensis was evaluated on the candidacidal activity, H2O2 and nitric oxide (NO) production, and expression of mannose receptors by murine peritoneal macrophages. Mice received three intraperitoneal (i.p.) injections of ATF and after 48 h their peritoneal resident macrophages were assayed against Candida albicans yeast forms. The treatment increased fungicidal activity and it was associated with higher levels of H2O2, whereas NO production was not affected. We also found that the treatment enhances mannose receptor expression by peritoneal macrophages, which are involved in the attachment and phagocytosis of non-opsonized microorganisms. Treatment of animals with ATF was able to enhance the clearance of C. albicans during the first 6 h after the experimental i.p. infection. Our results suggest that this extract can increase host resistance against some infectious agents through the stimulation of microbicidal activity of macrophages.

Exporting "failure": why research from rich countries may not benefit the developing world

The '10/90 gap' was first highlighted by the Global Forum for Health Research. It refers to the finding that 90% of worldwide medical research expenditure is targeted at problems affecting only 10% of the world's population. Applying research results from the rich world to the problems of the poor may be a tempting, potentially easy and convenient solution for this gap. This paper had the objective of presenting arguments that such an approach runs the risk of exporting failure. Health interventions that are shown to be effective in the specific context of a Western industrialized setting will not necessarily work in the developing world.

Detection of circulating polysaccharide antigen of Schistosoma mansoni in hamster sera by crossed immunoelectrophoresis

Crossed immunoelectrophoresis (IEC) was used for detection of free and complexed circulating polisaccharide anodic antigen (AgCA) of Schistosoma mansoni in sera of infected hamsters. An attempt was also done to detect AgCA in human sera from patients infected with S. mansoni. The conditions for isolation and detection of complexed AgCA were established. The sensitivity of IEC was increased by incorporation of 2% polyethylene glicol (PEG) to the agarose and by maintaining the system at 4°C during the electrophoretic run. Free AgCA was detected in 12 and the complexed in 30 of the 37 hamsters sera analysed. Correlation between AgCA (free and complexed) and the parasite load was observed. AgCA was not detected, under the experimental conditions used, in human sera from 7 patients in the acute and 23 in the chronic phase of infection.

Schistosoma mansoni circulating polysaccharide and protein antigens recognized by sheep antisera in patients with different clinical forms of schistosomiasis before and after treatment

Two sheep antisera, one of which raised against polysaccharide (Po) and other against protein (Pt) components of Schistosoma mansoni adult worms, were assessed by ELISA for their ability to detect circulating parasite antigens in patients with different clinical forms of chronic schistosomiasis mansoni. The former antiserum detected parasite antigens in liver granulomata and the latter in renal glomeruli from schistosomiasis patients and mice experimentally infected with S. mansoni. In general, the levels and/or positivity rate of circulating antigens and specific IgG antibodies were significantly higher in patients with hepatointestinal (HI) and hepatosplenic (HS) forms than in mild intestinal (I) forms. An association between Po antigens and clinical features of the disease was observed, as the level of these antigens was low (137 ng/ml) as well as the positivity rate (7.9%) in patients with I forms; values that were intermediate (593 ng/ml and 33.3%) in those with HI forms, and high (1.563 ng/ml and 50.0%) in more severe HS forms. The Pt antigens were detected in the studied clinical forms not differing statistically but, the positivity rate was significantly higher in HS forms comparatively to I forms. The antisera studied revealed distinct circulating antigen profiles, and the prognostic value of Po and Pt antigens was suggested.

Dot-enzyme-linked immunosorbent assay (Dot-ELISA) for detection of pneumococcal polysaccharide antigens in pleural fluid effusion samples.: Comparison with bacterial culture, counterimmunoelectrophoresis and latex agglutination

A dot-enzyme-linked immunosorbent assay (Dot-ELISA) for pneumococcal antigen detection was standardized in view of the need for a rapid and accurate immunodiagnosis of acute pneumococcal pneumonia. A total of 442 pleural fluid effusion samples (PFES) from children with clinical and laboratory diagnoses of acute bacterial pneumonia, plus 38 control PFES from tuberculosis patients and 20 negative control serum samples from healthy children were evaluated by Dot-ELISA. The samples were previously treated with 0.1 M EDTA pH 7.5 at 90°C for 10 min and dotted on nitrocellulose membrane. Pneumococcal omniserum diluted at 1:200 was employed in this assay for antigen detection. When compared with standard bacterial culture, counterimmunoelectrophoresis and latex agglutination techniques, the Dot-ELISA results showed relative indices of 0.940 to sensitivity, 0.830 to specificity and 0.760 to agreement. Pneumococcal omniserum proved to be an optimal polyvalent antiserum for the detection of pneumococcal antigen by Dot-ELISA. Dot-ELISA proved to be a practical alternative technique for the diagnosis of pneumococcal pneumonia.

Detection of Cryptococcus neoformans capsular polysaccharide antigen in asymptomatic HIV-infected patients

Serum samples from 242 HIV-positive persons were studied for the detection of capsular polysaccha-ride antigen of Cryptococcus neoformans; 193 of these patients presented less than 300 CD4+ cells/µl of blood and 49 patients had more than 300 CD4+ cells/µl. None of them had symptoms or signs characteristic of cryptococcosis. The capsular antigen of C. neofarmans was detected by latex agglutination technique with pronase pre-treatment (IMMY, Crypto-Latex Antigen Detection System, Immunomycologics Inc., OK, USA); in 61% of the samples, ELISA technique was also used (Premier, Cryptococcal Antigen, Meridian Diagnostic Inc., Cincinatti, Oh, USA). The comparative study of both methods showed that the results obtained were similar in 96.9% of the cases. The capsular antigen was detected in 13 out of 193 (6.7%) patients with less than 300 CD4+ cells/µl. Cryptococcosis was confirmed mycologically in 3 of these 13 cases (23%) by the isolation of C. neoformans in CSF or blood cultures. Three patients, who had presented negative results of both tests for capsular antigen, suffered disseminated cryptococcosis 4 to 8 months later. The predictive diagnostic value of capsular antigen detection of C. neoformans seems tobe low and we believe that it should not be done routinely in asymptomatic HIV-positive persons.

Histoplasmin reaction. Comparison of a polysaccharide antigen to the filtrate antigen

This work was planned by taking into account all the knowledge accumulated from the immunological study of paracoccidioidomycosis. It aimed at comparing a polysaccharide antigen from Histoplasma capsulatum to a classic histoplasmin with the help of intradermal tests of delayed type of hypersensitivity. Tests were applied to 115 individuals in Santo Amaro, a town in the state of São Paulo. Positive results using classic histoplasmin were obtained in 46.0% cases whereas positive results using the polysaccharide antigen at its hightest concentration were obtained in 51.30% cases. The major conclusion in this investigation is that it is possible to use the polysaccharide antigen as histoplasmin instead of the filtrate antigen

The FML (Fucose Mannose Ligand) of Leishmania donovani: a new tool in diagnosis, prognosis, transfusional control and vaccination against human kala-azar

The Fucose-Mannose Ligand (FML) of Leishmania donovani is a complex glycoproteic fraction. Its potential use as a tool for diagnosis of human visceral leishmaniasis was tested with human sera from Natal, Rio Grande do Norte, Brazil. The FML-ELISA test, showed 100% sensitivity and 96% specificity, identifying patients with overt kala-azar (p < 0.001, when compared to normal sera), and subjects with subclinical infection. More than 20% apparently healthy subjects with positive reaction to FML developed overt kala-azar during the following 10 months. In the screening of human blood donnors, a prevalence of 5% of sororeactive subjects was detected, attaining 17% in a single day. The GP36 glycoprotein of FHL is specifically reconized by human kala-azar sera. The immunoprotective effect of FML on experimental L. donovanii infection was tested in swiss albino mice. The protection scheemes included three weekly doses of FML, supplemented or not with saponin by the subcutaneous or intraperitoneal routes and challenge with 2x 10(7) amastigotes of Leishmania donovani. An enhancement of 80.0 % in antibody response (p<0.001) and reduction of 85.5 % parasite liver burden (p<0.001) was detected in animals immunized with FML saponin, unrespectivety of the immunization route.

Carbohydrate-rich high-molecular-mass antigens are strongly recognized during experimental Histoplasma capsulatum infection

INTRODUCTION: During histoplasmosis, Histoplasma capsulatum soluble antigens (CFAg) can be naturally released by yeast cells. Because CFAg can be specifically targeted during infection, in the present study we investigated CFAg release in experimental murine histoplasmosis, and evaluated the host humoral immune response against high-molecular-mass antigens (hMMAg. >150 kDa), the more immunogenic CFAg fraction. METHODS: Mice were infected with 2.2x10(4) H. capsulatum IMT/HC128 yeast cells. The soluble CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg circulating immune complexes (CIC) levels were determined by enzymelinked immunosorbent assay, at days 0, 7, 14, and 28 post-infection. RESULTS: We observed a progressive increase in circulating levels of CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg CIC after H. capsulatum infection. The hMMAg showed a high percentage of carbohydrates and at least two main immunogenic components. CONCLUSIONS: We verified for the first time that hMMAg from H. capsulatum IMT/HC128 strain induce humoral immune response and lead to CIC formation during experimental histoplasmosis.

COMPORTAMENTO FENOLÓGICO DA SUCUPIRA-PRETA (Diplotropis purpurea (Rich.) Amsh. var. coriacea Amsh.), NA RESERVA FLORESTAL DUCKE

Este trabalho analisa dados de 6 anos (1980-1985) de observações da fenologia de cinco indivíduos arbóreos da espécie Diplotropis purpures (Rich.) Amsh. var. coriacea Amsh., da família Leguminosae, subfamília Papilionoideae, localizados na Reserva Florestal Ducke. Determinou-se a época, duração e freqüência das fases reprodutivas, bem como o tipo de mudança foliar. A espécie apresentou a fase de floração na estação seca. A fase de frutificação foi observada no meio da estação seca e início da estação chuvosa. A duração média da fase de floração foi de 6 meses e a fase de frutificação 7 meses. Ao nível de espécie, o padrão de ocorrência da fase de floração e frutificação foi anual à irregular. Porém, ao nível de indivíduo, o padrão de comportamento foi irregular. Quanto ao tipo de mudança foliar, a espécie apresentou características de ser semi-caducifólia durante a floração, na época seca.

Atividade antimicrobiana de fungos endofíticos isolados de plantas tóxicas da amazônia: Palicourea longiflora (aubl.) rich e Strychnos cogens bentham

Das plantas tóxicas da Amazônia Palicourea longiflora e Strychnos cogens foram isolados 571 fungos endofíticos e 74 bactérias endofíticas. Palicourea longiflora (Rubiaceae) e outras espécies desse gênero estão relacionadas a 90% das mortes de gado na região Amazônica. Strychnos cogens (Loganiaceae) e outras espécies de Strychnos são utilizadas pelos indígenas na confecção de "curares". Entre os endófitos isolados de P. longiflora foram identificados os fungos: Colletotrichum sp. e seu telemorfo Glomerella sp., Guignardia sp., Aspergillus niger, Phomopsis sp. e Xylaria sp., além da bactéria Burkholderia gladioli, pertencente a um grupo de fixadoras de nitrogênio. Dos isolados de S. cogens foram identificados os fungos: Colletotrichum sp., Guignardia sp., Aspergillus niger e Trichoderma sp. Uma amostra de 79 isolados fúngicos teve seus metabólitos extracelulares bioensaiados contra microrganismos patogênicos e fitopatogênicos: 19 isolados inibiram um ou mais microrganismos-teste. Dos oito isolados com melhores resultados de inibição, as móleculas bioativas eram menores que 12.000 daltons, fato verificado pela diálise dos metabólitos.

Superação de dormência na qualidade de sementes e mudas: influência na produção de Senna multijuga (L. C. Rich.) Irwin & Barneby

O objetivo deste trabalho foi determinar a qualidade fisiológica e sanitária de sementes de Senna multijuga(L. C. Rich.) Irwin & Barneby relacionada aos métodos de superação de dormência e à interferência na produção de mudas. As sementes foram submetidas aos seguintes métodos: imersão em água fervente, as sementes foram imersas em água, com temperatura de 100°C, até esfriar, por 24 horas; escarificação ácida, onde as sementes foram imersas em ácido sulfúrico (H2SO4) a 90%, por 10 e 20 minutos, e testemunha (sem tratamento). Foram realizados os testes de sanidade, germinação, tetrazólio e avaliação da qualidade das mudas. O delineamento experimental foi inteiramente casualizado. Para a avaliação da germinação foi utilizado um esquema fatorial (4 X 2), com quatro métodos de superação de dormência X dois fotoperíodos, para os substratos rolo-de-papel e vermiculita. A escarificação ácida constituiu-se no método mais eficiente para a superação da dormência das sementes de Senna multijuga. Penicillium sp. e Aspergillus sp. tiveram sua incidência aumentada quando o tegumento foi danificado pela escarificação ácida por 20 minutos. O controle de Fusarium spp. aumentou gradativamente com o aumento do tempo de exposição ao ácido sulfúrico.

As condições ecológicas da Cephaelis ipecacuanha Rich

The present paper is a simple introduction to the ecological requirements of Cephaelis ipecacuanha and only a few preliminary conclusions are reached. Cephaelis ipecacuanha has the habit of a forest plant, which is closely connected with the meteorological and floristic factors. This makes it very necessary to study its ecolo-gical relationships carefully, so as to understand its productivity in accordance with its mi¬croclimatic and microedaphic reactions. Conclusions: 1 — Within the ecological range of Cephaelis ipecacuanha, some zones show more fa¬vourable phytosociological conditions than other zones. Consequently, the vegetative and biological types must be investigated in different associations. a — The most favourable vegetative and biological types encountered, in regard to the phytosociological characteristcs of Cephaelis ipecacuanha, were found respectively in the rain forest on the slopes of the Serra dos Parecis and in the serclimax of the river Galera and its tributaries. b — The phytosociological optimum of Cephaelis ipecacuanha was seen in the Vochysietum (Vochysia sp.) which corresponds to number 11 of the original text. It can be defined as follows: shading by vegetation approximately 90%, as the herbaceous sinusium covers 90% of the area worked in, though the arboreal and arbustive coverture is only 65% and 60% respectviely. The inclination is pratically nil (less than 5°) but the drainage is good because the soil is deep and silicous-humous, thus facilitating infiltration by the rain water which inundates the ground temporarily during the periods of floods. The pH oscillates between 5 and 6, denoting a slightly acid soil.